Multiple monolayers of polymeric linkages on a solid phase for immobilizing macromolecules

ABSTRACT

The present invention provides a process for building an immobilized structure comprising multiple monolayers of effective sequential polymeric linkages. The multiple monolayers are built, monolayer by monolayer, from the surface of a solid phase, by an alternating reaction sequence conducted with multifunctional reagents. The process provides the capability of building numerous immobilized multiple monolayer structures wherein the length of each structure extending from a solid phase is substantially the same. Structures comprising multiple monolayers of polymeric linkages are useful for many purposes, as for example, immobilizing macromolecules or other biomolecules and the like to produce chemical or biochemical sensors. Other uses extend to molecular electronic applications, such as the production of molecular conductive wires and molecular circuitry. In a preferred embodiment, carbonyldiimidazole and phenylenediamine are the multifunctional reagents and multiple monolayers of phenylurethane linkages are produced.

This application is a continuation, of application Ser. No. 16,337,filed 10/07/86 now abandoned.

FIELD OF THE INVENTION

This invention relates to a process for building multiple monolayers ofpolymeric linkages wherein the length of each of the resulting multiplemonolayer structures can be controlled so that they may be substantiallythe same. Structures comprising multiple monolayers of polymericlinkages are useful for many purposes, as for example chemical orbiochemical sensors, molecular conductive wires, and the like.

BACKGROUND OF THE INVENTION

The assembling of molecular layered structures has gained recognition inthe past, primarily in the context of solid phase synthesis of peptides.R. B. Merrifield reports in Biochemistry, Vol. 3, No. 9 pp. 1385-1390(1964), the stepwise synthesis of the naturally occurring nonapeptidebradykinin, by attachment of a terminal amino acid to a substrate, adecarboxylation step, and then a coupling step wherein an amino acidresidue is attached to the terminal amino acid. The steps are repeateduntil a peptide with nine amino acid residues is produced.

More recently, this approach to protein synthesis has been expanded tothe production of a multilayer film of 1,5-hexadecenyl-trichlorosilane.Lucy Metzer and Jacob Sagiv in J. Am. Chem. Soc., Vol. 105, pp. 674-676(1983) report a two-step sequence consisting of monolayer absorption,followed by chemical activation of the exposed surface to provide polarabsorption sites for the anchoring of the next monolayer.

SUMMARY OF THE INVENTION

The present invention provides a process for building an immobilizedstructure comprising multiple monolayers of "effective sequentialpolymeric linkages," said process comprising the steps of:

(a) providing a solid phase with a first attached "reactive moiety";

(b) conducting a first coupling reaction by reacting said attached"reactive moiety" with an excess of a first multifunctional reagent orcombination of reagents, said reagent or combination of reagentscomprising a first functional group or groups reactive with said firstreactive moiety and a second functional group or groups reactive with asecond multifunctional reagent or combination of reagents; said couplingreaction producing a first intermediate adduct derived from said firstreagents and attached to said solid phase, said adduct comprising aresidue and a second reactive moiety capable of coupling to the secondmultifunctional reagent;

(c) conducting a second coupling reaction by reacting said firstintermediate adduct with an excess of the second multifunctionalreagent, to form a first molecular unit comprising a residue derivedfrom said first intermediate adduct and a residue from said secondreagent, said molecular unit capable of reacting with said firstmultifunctional reagent;

(d) repeating said first coupling and second coupling reactionsalternatively to produce an immobilized structure comprising multiplemonolayers of "effective sequential polymeric linkages".

The process provides the capability of building numerous immobilizedmultiple monolayer structures wherein the length of each structureextending from the substrate is substantially the same.

Another aspect of the invention relates to the immobilized structuresprepared in accordance with the process.

"Effective sequential polymeric linkages" within the context of theinvention are at least two molecular units, sequentially bonded,preferably having either conjugated moieties capable of acting asconductive or semiconductive molecular entities through a pi (π) bonddelocalization; or unconjugated moieties that are not conductive, andthus capable of acting as resistive molecular entities. In either of thepreferred cases, biomolecules or other macromolecules and the like mayalso be immobilized by physical or covalent bonding to terminalfunctional groups of the multiple monolayer structures.

The solid phase in the present invention is one that has a "reactivemoiety" available on a surface that is capable of reacting with thefirst multifunctional reagent and incorporating at least a part of thatreagent to form an immobilized intermediate adduct having a secondreactive moiety capable of reacting with the second multifunctionalreagent.

In the context of the invention, a "reactive moiety" is a molecularmoiety that has the capability of reacting with another moiety viaclassical functional group organic chemistry. Of these may be mentionedsuch moieties as amino groups, carboxylic acids, carbonylimidazoles,aldehydes, carboxyacid halides, carboxylated esters, carboxylatedanhydrides, alcohols or phenols, and the like; of which amino groups andcarbonylimidazoles are preferred due to their high reactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the radioisotopic data obtained after each completereaction cycle adding a successive monolayer of phenylurethane.

FIG. 2 is a plot of immobilized acetylcholinesterase activities after a6-month period of time relative to bond lengths of phenylurethanelinkages.

FIG. 3 is a plot of the change in bond lengths of an immobilizingmonolayer structure relative to percent enzyme activity of the enzymeimmobilized with the multiple monolayer structure.

DETAILED DESCRIPTION OF THE INVENTION

In the process of the present invention, an immobilized structurecomprising multiple monolayers of effective sequential polymericlinkages is built, monolayer by monolayer, from the surface of a solidphase, by an alternating reaction sequence conducted withmultifunctional reagents.

According to the method of the invention, a solid phase with a firstattached reactive moiety is contacted with a first multifunctionalreagent or combination of reagents. The method of contact may be by anymeans that allows the reactants to come into contact with the solidphase.

For example, the substrate containing the reactive moiety may he reactedwith the chosen first multifunctional reagent or combination of reagentsin a conventional manner in the vapor state or by simply mixing thereactants together in a liquid state. When the reaction is carried outin the liquid state, conventional solvents are often employed. In somecases, the multifunctional reagent may be susceptible to hydrolysis, andthus, an aprotic solvent should be employed It is generally preferred toemploy an excess of the multifunctional reagent to avoid bimoleculartermination reactions which produce products on the solid phase whichwill interfere with the desired building of the polymeric monolayers.

Successful completion of the first coupling reaction will result in anintermediate adduct containing at least a part of the first reagent, theadduct terminating with at least one second reactive moiety. At thistime it is often preferred to employ an additional step of removingunreacted first multifunctional reagents, usually by conventionalwashing techniques.

In the second coupling step of the present method, the substrate nowhaving the intermediate adduct affixed to it is reacted in a similarfashion with a second multifunctional reagent or a combination thereof.During this step, at least a portion of the second reagent orcombination of reagents is incorporated, producing one monolayer of thedesired polymeric linkage, this monolayer terminating with at least onemoiety capable of reacting with the first multifunctional reagent, andthus repeating the cycle to produce a second monolayer, and so on. Thereaction cycles may be repeated in this alternate manner to produce thedesired number of monolayers, with the terminal end of the overallstructure ending with at least one reactive moiety belonging to eitherone of the two types of reactive moieties generated.

Solid phases having reactive moieties attached to their surfaces usefulin the practice of the invention can be any solid material that can actas a substrate for attachment of the multiple monolayers, and serves toanchor the reaction products to a solid phase and permit the unreactedmultifunctional reagents in each step to be efficiently removed prior tothe next reactive step with another multifunctional reagent. Selectionof these substrates may be governed in part by their physical andchemical properties, such as solubility, functional groups, mechanicalstability, surface area swelling propensity, hydrophobic or hydrophilicproperties, and the like; as well as their electrical properties, suchas conductivity, resistivity, and the like.

Essentially three major types of substrates are most preferred:inorganics, natural polymers, and synthetic polymers. Of these may bementioned polymers such as polyvinylalcohols, acrylates and acrylicacids such as polyethylene-co-acrylic acid, polyethylene-co-methacrylicacid, polyethylene-co-ethylacrylate, polyethylene-co-methyl acrylate,polypropylene-co-acrylic acid, polypropylene-co-methyl-acrylic acid,polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate,polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and thelike; and those containing acid anhydride groups such aspolyethylene-co-maleic anhydride, polypropylene-co-maleic anhydride andthe like; and natural polymers such as polypeptides, proteins andcarbohydrates; metalloids that have semiconductive properties such assilicon, germanium, aluminum, and the like; and metals such as platinum,gold, nickel, copper, zinc, tin, palladium, silver. Particularly usefulare oxides of the metal and metalloids such as Pt-PtO, Si-SiO, Au-AuO,TiO₂, Cu-CuO, and the like. Inorganics having conductive orsemiconductive properties are preferred in some embodiments of theinvention. Of these may be mentioned silicon/silicon oxide semiconductorelements, or other semiconductor elements such as lithium niobate,galluim arsenide, indium phosphide, and the like.

Substrates such as those mentioned above, particularly the metalloids,may be treated to contain an appropriate reactive moiety or in somecases may be obtained commercially already containing the reactivemoiety. Materials containing reactive surface moieties such as aminosilane linkages, hydroxyl linkages, or carboxysilane linkages arepreferred in some embodiments of the invention and may be produced bywell established surface chemistry techniques involving silanizationreactions, or the like. Examples of these materials are those havingsurface silicon oxide moieties, covalently linked togamma-aminopropylsilane, and other organic moieties;N-[3-(triethyoxysilyl) Propyl]Phthelamic acid; andBis-(2-Hydroxyethyl)aminopropyltriethoxysilane. Exemplary of readilyavailable materials containing NH₂ reactive functionalities arepara-aminophenyltriethyoxysilane and gamma-aminopropyl silanizedControlled Porous Glass Beads, available from Pierce Chemical Co., P.O.Box 117, Rockford, Ill. 61105.

The multifunctional reagents useful in the process of the invention arethose that can react in alternate manner to produce at least onemolecular unit of the desired effective polymeric linkage, the alternatereaction scheme capable of repetition until the desired number ofmolecular units are produced. Combinations of first and secondmultifunctional reagents may be varied to obtain a wide variety ofpolymeric linkages with electrical properties ranging from nonconductiveto semiconductive and conductive. Terminal functional groups of theresulting multiple monolayer structure may be further reacted withbiomolecules, other macromolecules, and the like. Combinations of firstmultifunctional agents may also be used in conjunction with combinationsof second multifunctional reagents to produce branched polymericlinkages and the like.

In general, at least a part of the first multifunctional reagent orcombination of reagents is incorporated into the immobilized molecularunit, producing an intermediate adduct with at least one terminalfunctional group that is capable of reacting with the secondmultifunctional reagent. Upon reaction with the second multifunctionalreagent or combination of reagents, at least a portion of that secondreagent is incorporated into the immobilized structure, producing onecomplete molecular unit with at least one terminal group capable ofreacting with the first multifunctional reagent. The cycle may then berepeated. It is generally preferred that the terminal group or groups ofa completed molecular unit be the same reactive moiety as thatoriginally provided on the substrate.

The first multifunctional reagent or combination of reagents in thepresent process is selected according to the type of multiple monolayerstructure it is desired to achieve, and may be heterofunctional orhomofunctional. The first reagent or combination thereof is capable ofreacting with the reactive moiety attached to the solid phase.

Illustrative of multifunctional reagents suitable for use as the firstreagent whether alone or in combination may be mentioned those thatreact with the substrate moiety via nucleophiliac displacement and maybe represented by the following general formula I: ##STR1## wherein R isreadily displaced by nucleophiles, and is preferably aromaticheterocyclic or halogen.

A further illustration of reagents suitable as the first multifunctionalreagents are those capable of reacting with the substrate reactivemoiety via a condensation/elimination scheme, and may be represented bythe general formulae II and III: ##STR2## wherein R₁ is phenyl,polyphenyl, fused ring aromatic, alkyl, alkene, aromatic heterocyclic,metalloorganic; R₂ is H, halogen, OR₄, OH, SH; and R₃ is H, halogen,OR₄, OH, or SH, wherein R₄ is an aliphatic or aromatic hydrocarbon; and##STR3## wherein R₁ is phenyl, polyphenyl, fused ring aromatic, alkyl,alkene, aromatic heterocyclic, metalloorganic; R₂ --N-- is HO--N--,O═N--, X⁻ N₂ +--; R₃ --N-- is HO--N--, O═N--, X⁻ N₂ ⁺ --.

The second multifunctional reagent is one that will react with theterminal functional group or groups of the intermediate adduct producedby the first reaction.

The second multifunctional reagent or reagents may be heterofunctionalor homofunctional and are selected from the group of reagentsrepresented by the following formula IV: ##STR4## wherein R is phenyl,polyphenyl, fused ring aromatic, alkyl, alkene, aromatic hetercyclic, ormetalloorganic; X₁ and X₂ are the same or different and are NH₂, NHOH,or OH; X₃ is NH₂, NHOH, OH, or H; and X₄ is NH₂, NHOH, OH or H.

First and second reagents are chosen according to the polymeric linkagesit is desired to build. When first multifunctional reagents are selectedfrom general formula I, the second multifunctional reagents should beselected to contain nucleophilic groups that will react with theterminal functional moiety or moieties of the intermediate adductproduced from the first coupling reaction, via a further displacementreaction. When the first reagents are selected from general formulae IIor III, the second multifunctional reagents should be ones that willreact via condensation/elimination. In general, reaction cyclesconducted with a combination of a first reagent acting via nucleophilicdisplacement with a conjugated second reagent will producesemiconjugated polymeric linkages with potential semiconductivity, whilereaction with unconjugated reagents produces non-conjugated linkages.

Reaction cycles conducted with a first reagent acting viacondensation/elimination and a conjugated second reagent will produceconjugated polymeric linkages, potentially conductive. Reaction cyclesconducted with an unconjugated second reagents will producenon-conjugated linkages.

Typical schemes for utilizing the various first and second bifunctionalreagents are depicted in the following Table:

      TYPICAL SCHEMES FOR SOLID-PHASE SYNTHETIC LINKAGES  First Second  Solid     Bifunctional Bifunctional Phase Reagent Reagent Solid-Phase Synthetic     Linkage      ##STR5##      ##STR6##      ##STR7##      ##STR8##      ##STR9##      ##STR10##      ##STR11##      ##STR12##      ##STR13##      ##STR14##      ##STR15##      ##STR16##      ##STR17##      ##STR18##      ##STR19##      ##STR20##      ##STR21##      ##STR22##      ##STR23##      ##STR24##      ##STR25##      ##STR26##      H.sub.2      N(CH.sub.2).sub.xNH.sub.2     ##STR27##      ##STR28##      ##STR29##      ##STR30##      ##STR31##

The following presents a typical scheme for synthesis of a branchedmultiple monolayer structure wherein the polymer or oligomer produced ispotentially semiconductor and could be utilized as a two-dimensionalmolecular wire. ##STR32##

It should be appreciated that various combinations of multiple monolayerunits with varying electrical properties could be combined to formvarious electrical component parts on a molecular level. For example,electrical "switches", "resistors" and the like are within thecontemplation of the invention. The following presents a typical schemefor formation of a molecular switch: ##STR33##

As demonstrated above, semiconjugated or conjugated linkages, in thiscase, polybenzoiimines, are provided on conductive substrates with acompound containing a displaceable metal, capable of acting as amolecular switch, interposed between portions of the linkages. When asufficient voltage or other stimulus is applied across this structure,the metal will be displaced from the molecular switch, allowing electriccurrent to be conducted. The first polymeric linkages may be provided onthe substrate according to the process of the invention, generating aterminal reactive group capable of reacting with the metalloporphyrin.Additional polymeric monolayers are then built, monolayer by monolayer,from the metalloporphyrin molecular switch, and the terminal ends of thesecond linkages are then reacted with reactive moieties on the secondsubstrate for attachment thereto.

Compounds suitable for use as molecular switches are those that canreact to an applied voltage or other stimulus such as photoactivation orthe like, in such a way as to preclude or allow the passage ofelectrons. Useful in this regard are compounds such as themetalloporphyrins, phthalocyanines, hemiquinones, and the like.Plausible schemes also exist for incorporating charge-transfer saltssuch as TTF-TCNO (tetrathiafulvalene-tetracyanoquinodimethane). Forexample, diamino-TTF could be covalently coupled within the backbone ofa solid-phase synthesized conjugated polymer, and then TCNO moleculescould be allowed to chelate to the TTF units from solution to form theTTF-TCNO charge-transfer salt moieties at a specific position in themolecular circuit. For compounds that may act as molecular switches, seeBrian M. Hoffman and James A. Ibers, "Porphyrinic Molecular Metals,"Acc. Chem. Res. 1983, 16, 15-21; T. W. Barrett et al., "ElectricalConductivity in Phthalocyanines Modulated by Circularly PolarizedLight," Nature, vol. 301, 24 February, 1983; and, 2nd InternationalWorkshop on Molecular Electronic Devices, Naval Research Lab, WashingtonD.C., Apr. 13-15 (1983).

Unconjugated areas that essentially act as a molecular resistor could befurther combined with a molecular wire section and molecular switch tomodulate electric current passing through. A scheme for a simplemolecular circuit is presented below, wherein molecular conductive wiresof polybenzoiimines are conjugated to a metalloporphyrin switch inparallel with an n-propyldiamine resistor: ##STR34##

It should be appreciated that the stepwise monolayer addition of thepresent method can provide a substrate having numerous multiplemonolayer structures originating from its surface, with a narrowdistribution of total monolayer lengths. In some embodiments, theprecision obtained is within a few Angstrom units. This is particularlyuseful in the area of conductive or semiconductive polymers. Uniformityof oligomeric or polymeric lengths would be helpful in buildingmolecular level circuits and the like as depicted above, as carefulcontrol may be exerted over the length of each of the components makingup the circuit. Such uniformity is not easy to achieve with thetechniques presently available to the art.

It should also be appreciated that the number of multiple monolayerstructures prepared on the surface of a substrate will be governed bythe number of initial reactive sites on the substrate surface, as wellas other factors such as steric hindrances between the monolayerlinkages themselves. The number of initial sites could be such thatenough multiple monolayers may be prepared to form an actual layeracross all or a portion of the substrate surface, the thickness of thelayer governed by the number of reaction cycles completed, buildingsuccessive monolayers on each of the respective structures. The presentprocess thus also provides an alternative to conventional film formingtechniques.

Along these same lines, the spacing between the various multiplemonolayer structures can be controlled by adjusting the availability ofreactive moieties on the solid phase. In some embodiments of the presentinvention, spacing between linkages has been in the order ofapproximately 23 Angstrom units. In this case, the spacing could beincreased to approximately 50 Angstrom units by passivating theavailable reactive sites on the solid phase by approximately 50%. In apreferred embodiment wherein multiple monolayers of phenylurethanelinkages are produced, the spacing of NH₂ groups on the solid phase maybe altered by passivating approximately 50% of the available sites withdichlorodimethylsilane prior to the monolayer synthesis procedure. Thus,the linkages could be packed at approximately 50 Angstrom centers, whichmight be advantageous in molecular electronic applications and the like.

Electroactive properties of conductive or semiconductive polymersprepared according to the above reaction schemes may be enhanced throughthe use of conventional p or n doping. For example, dopants such as I₂,conventionally used to dope polyacetylenes, could be used to dope theconjugated linkages produced by this methodology.

After the desired number of monolayers of a particular molecular unit isprepared, the terminal end of the multiple monolayer structure may befurther reacted with a variety of molecules. Useful in this regard aremacromolecules such as chelated crown ethers, or biomolecules such asenzymes, hormones, antigens, antibodies, membrane receptors and thelike. Immobilization of biomolecules through the use of the immobilizedmonolayer structure enhances the stability of the biomolecules and thusis an alternative to direct immobilization to a substrate. In someembodiments, the stability of a particular enzyme is directlyproportional to the length of the multiple monolayer structure. Greaterthan about four monolayers is generally preferred, with greater thanabout ten monolayers particularly preferred. One skilled in the artshould be able to readily optimize the stability of a biomolecule ofinterest by testing its stability relative to various monolayer lengths.

It is preferable that covalent bonding techniques be employed whenimmobilizing biomolecules. Conventional techniques that are applicablehave been described in the art. One such treatise is "ImmobilizedEnzymes, Antigens, Antibodies, and Peptides, Preparation andCharacterization," edited by Howard H. Westall, Marcel Dekker, Inc.(1975).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following presents a preferred reaction scheme according to thepresent invention for production of a multiple monolayer structurecomprised of electroactive polyphenylurethane linkages: ##STR35##

Substrate containing a reactive moiety, preferably NH₂, is reacted firstwith carbonyldiimidazole in a solvent. Solvents particularly useful inthis regard are aprotic, so as to avoid the side reaction of hydrolysisof the carbonyldiimidazole. Of the solvents that may be mentioned aspreferable are dimethylformamide, dimethylsulfoxide, benzene,chloroform, acetone, ether, and the like.

Amounts of carbonyldiimidazole employed in this first coupling step mayvary widely, and should be an amount sufficient to avoid the productionof termination reaction products which will interfere with the desiredbuilding of the polymeric monolayers. For this reason, an excess of thereagent may generally be employed. For example, for 1 milliequivalent ofamino moieties, amounts of about 1 milliequivalent to greater than about10 milliequivalents of carbonyldiimidazole can be employed, with about 6milliequivalents to 10 milliequivalents more preferable. The couplingreaction may be carried out by conventional techniques, such as bysimply mixing the reactants together. The reaction time period may varywidely, and generally ranges from about 15 minutes to about 60 minutes.The temperature at which the reaction takes place may also vary widelyand is generally carried out at room temperature for the sake ofconvenience.

It is preferable to employ an additional step of removing unreactedcarbonyldiimidazole. This can usually be accomplished by conventionalfiltering and washing techniques, preferably by washing with an aqueoussolution and then an aprotic solvent to decompose unreactedcarbonyldiimidazole and remove residual products from the solid phase.

The above coupling reaction will produce an imidazole derivativeattached to the substrate, which is reactive to a second multifunctionalreagent, preferably a diamine such as 1,4 phenylenediamine. Here again,it is preferable to employ an excess of this second reagent to minimizebimolecular termination reactions which can produce products on thesolid-phase which will interfere with the desired building of thepolymeric monolayers.

Contact of the imidazole derivative with the second reagent is effectedby conventional techniques. Contact time periods vary widely, with atleast about 15 minutes suitable for the reaction.

Completion of the second coupling reaction produces one monolayer ofphenylurethane. The alternative reaction scheme may then be repeated anumber of times to produce the desired length of the overall structure,the terminal group ending with an amino functionality orhemi-carbonyldiimidazole functionality.

In particularly preferred embodiments, the thus produced multiplemonolayer structure is used to immobilize certain enzymes or otherproteins and the like. The immobilization may take place usingconventional techniques. For example, contact of the firstmultifunctional reagent with the terminal moiety of the multiplemonolayer structure will produce the intermediate adduct that in turnmay react with amino groups contained in the enzyme. Thus, covalentbonding of the enzyme to the monolayer structure is achieved.

Illustrative of the many uses of the immobilized multiple monolayerstructures as described hereinabove may be mentioned applications in thearea of biological and chemical sensors. Any number of antibodies,enzymes, hormones, membrane receptors, or the like can be attached tothe multiple monolayer structures, to produce the optimum moleculardesign for a particular sensing unit. In some embodiments of the presentinvention, the stability of a particular immobilized enzyme can beincreased manyfold by increasing the distance between the substrate andthe enzyme through the use of the multiple monolayer structure.

In addition, molecular wires may be produced by building and varyingwith a precision of within a few Angstrom units, the bond lengths ofelectroactive linkages attached to electrode and semiconductor surfaces.Along these same lines, combinations of these monolayer structures withcompounds acting as switches may be useful in making molecular scaleelectronic circuits.

The following examples depict specific embodiments of the presentinvention but are not to be considered limitive thereof.

EXAMPLE I Preparation of Multiple Monolayer Structures Consisting ofPolyphenylurethane Linkages:

1 gram of Controlled Porous Glass Beads (CPG) was washed with 4-5volumes of dry dimethylformamide (DMF) on a sintered glass funnel, thentransferred to a flask and 5.0 ml 0.25M carbonyldiimidazole (CDI) in DMFadded. The mixture was shaken for 15 minutes at room temp, then filteredand washed with water, and 4-5 volumes of DMF. The CDI treated CPG wastransferred to a flask and 5.0 ml 0.25M¹⁴ C p-phenylene diamine wasadded (100-500 DPM/micromole diamine) and shaken for 80 minutes. Themixture was filtered and washed with DMF until no ¹⁴ C was evident infiltrate.

The process was repeated up to 18 cycles. A sample from each cycle wascounted for ¹⁴ C and analyzed for NH₂ end groups. Radioisotopic dataindicated that for each complete reaction sequence, 1 monolayer wassuccessfully added to the structure, with a correlation coefficient of0.94. For a build-up of the first (8) monolayers, a similar correlationcoefficient of 0.9932 was obtained (see FIG. 1).

The surface area per gram of CPG beads was known to be approximately 70m² /gram. By using routine calculation procedures, it was possible tocalculate that the packing density of the phenylurethane linkages was anominal one-linkage/528A², with a 23A interspacial distance betweenadjacent linkages.

CPG--Pierce 23909 controlled pore glass, aminopropyl

DMF--Pierce 20673 Dimethylformamide

p-Phenylenediamine--Aldrich P-2,396-2 recrystallized from toluene mp143-144

¹⁴ C p-Phenylenediamine - Pathfinder 15.03 Hc/micromole

CDI--Pierce 20220--N,N¹ - carbonyldiimidazole

EXAMPLE II Acetylcholinesterase Immobilization & Stability Studies

Using the aforementioned series of glass beads with variable bondlengths of phenylurethane linkages, eel-type acetylcholinesterase wasimmobilized to the amino groups at the ends of these linkages withcarbonyl diimidazole (CDI). The immobilized enzyme-glass beads werestored in a refrigerator (ca. 10-deg. C.). Periodic assays were carriedout to determine how much of the initial enzyme activity was retainedover a period of six months. The data are summarized below:

    ______________________________________                                        Bond Lengths Versus Percent Activity of AChE Enzyme                           ______________________________________                                        No. of Monolayers 2     4        6   8                                        Bond Length (Angstroms)                                                                         21    35       49  60 A                                     Percent Activity  30    50       60  65%                                      After Six Months                                                              ______________________________________                                    

The effect of immobilizing bond length on enzyme stability is verypronounced. After six months, the shortest linkage (21 Å) retained 30%of enzyme activity versus 65% activity for the longest linkage (60 Å),an increase greater than twofold (see FIG. 2).

It should be appreciated that there is a mathematical correlationbetween the immobilizing bond length and long-term stability of theimmobilized enzyme. In fact, the correlation between the percent ofenzyme activity values and the logarithms of the immobilizing bondlengths (or number of monolayers) is a linear relationship. In anotherdata manipulation, the percent enzyme activity was plotted against thepercent change in total bond length per successive monolayer ofphenylurethane. This plot is very linear with a statistical correlationcoefficient of 0.9992 (FIG. 3).

What is claimed is:
 1. A process for building an immobilized structurecomprising multiple monolayers of sequential polymeric linkages, saidprocess comprising the steps of:(a) providing a solid phase with a firstattached reactive moiety; (b) conducting a first coupling reaction byreacting said attached reactive moiety with an excess ofcarbonyldiimidazole as a first multifunctional reagent to produce anintermediate adduct attached to said solid phase which comprises aresidue from said first reagent, and which provides a second reactivemoiety capable of coupling to the second multifunctional reagent; (c)conducting a second coupling reaction by reacting said intermediateadduct with an excess of phenylenediamine as a second multifunctionalreagent to produce a first monolayer of a phenylurethane molecular unitas said polymer linkage; said molecular unit providing a terminalreactive moiety capable of coupling to said first multifunctionalreagent: (d) repeating said first coupling and second coupling reactionsalternatively to produce additional monolayers of said phenylurethanemolecular unit to obtain said immobilized structure comprising multiplemonolayers of sequential polymeric linkage.
 2. The process of claim 1wherein said solid phase contains an amino functionality.
 3. A processfor immobilizing an enzyme comprising the steps of:(a) providing a solidphase with an attached amino functionality; (b) conducting a firstcoupling reaction by reacting said attached amino functionality with anexcess of carbonyldiimidazole to produce an intermediate adduct whichcomprises a carbonyl-containing residue derived from saidcarbonyldiimidazole, and a reactive moiety capable of coupling to asecond multifunctional reagent containing at least one aminofunctionality; (c) conducting a second coupling reaction by reactingsaid intermediate adduct with an excess of phenylenediamine as saidsecond multifunctional reagent to produce a first monolayer of aphenylurethane molecular unit; (d) repeating said first coupling andsecond coupling reaction to produce at least four monolayers ofphenylurethane linkages and a terminal amino functionality; (e) reactingsaid terminal amino functionality with carbonyldiimidazole to produce aterminal intermediate adduct capable of coupling to an enzyme; (f)reacting said resulting intermediate adduct with an enzyme to produce anenzyme immobilized onto a multiple monolayer structure.
 4. The processof claim 3 wherein the enzyme is acetylcholinesterase.
 5. A multiplemonolayer structure produced according to the process of claim
 1. 6. Animmobilized enzyme produced according to the process of claim 12.